Invented in 1983 by Kary Mullis, the advent of Polymerase Chain Reaction (PCR) is recognized as one of the most important scientific developments of the twentieth century. PCR has revolutionized molecular biology through vastly extending the capability to identify and reproduce genetic materials such as DNA. Nowadays PCR is routinely practiced in medical and biological research laboratories for a variety of tasks, such as the detection of hereditary diseases, the identification of genetic fingerprints, the diagnosis of infectious diseases, the cloning of genes, paternity testing, and DNA computing. The method has been automated through the use of thermal stable DNA polymerases and a machine commonly referred to as “thermal cycler.”
The conventional thermal cycler has several intrinsic limitations. Typically a conventional thermal cycler contains a metal heating block to carry out the thermal cycling of reaction samples. Because the metal block has a large thermal mass and the sample vessels have low heat conductivity, cycling the required levels of temperature is inefficient. The ramp time of the conventional thermal cycler is generally not rapid enough and inevitably results in undesired non-specific amplification of the target sequences. Temperature overshoot or undershoot pass temperature set points during ramping also reduced the desired outcome during sample analysis. The suboptimal performance of a conventional thermal cycler is also due to the lack of thermal uniformity during cycling large sample sets, as widely acknowledged in the art. Furthermore, the conventional real-time thermal cycler system carries optical detection components that are bulky and expensive.
As an alternative to the heating block design, other methods also have shortcomings in speed and uniformity. For example, force air or liquid cyclers lack the temperature uniformity between sample wells due to the fact that fluid velocity and temperature is reduced across the multitude of DNA sample typically used in PCRs (Maltezos et al., U.S. Pat. No. 8,003,370). Also, PCR microfluidic chips can achieve up to 15° C./sec ramping rates due to the small sample volumes (Handique, U.S. Pat. No. 8,105,783). However, these lab-on-chip devices currently remain as custom solutions due to bio-incompatibility and surface chemistry issues that interfere with the polymerase activity. Moreover, constant temperature methods exist, e.g., LAMP and TMA, to eliminate the need for a thermal cycler. These isothermal methods, however, requires different polymerase and preparation protocols that are less familiar to many geneticists or molecular biologists.
Quintanar (U.S. Pat. No. 6,472,186) discloses a thermal cycler using pressurized gas flow. The patent describes a method in which a single-phase fluid performs the thermal cycles during the PCR process. The patent outlined the used of first and second heat transfer gases, under pressure, as a means of providing fast and uniform heat transfer to a plurality of samples. These forced convection methods using air only marginally improve performance of a PCR platform given that the heat transfer rates for gas is much lower than liquids or phase-changing fluids.
Heff (US Pat. Publ. No. 20070026444) discloses a thermal cycler by thermodynamic method. The application describes a method in which adiabatic work is performed on analyte samples inside the reaction vessel. One embodiment uses a piston to drive pressure and temperature cycles. Although technically sound, the weakness of the design is related to the difficulty of manufacturing sealed pistons that can operate in a reliable manner for the large pressures required. Also cross-contamination is likely to occur during handling and operating the movable piston inside the sample vessels. This drawback reduces the usability of an unfamiliar design and severely limits the commercialization appeal of the technology.
There thus remains a considerable need for an alternative thermal cycler technology. A desirable device would allow (a) rapid, precise and uniform transfer of heat to effect a more specific amplification reaction of nucleic acids; and/or (b) monitoring of the progress of the amplification reaction in real time; and/or (c) contaminant-free and user-friendly operation. The present invention satisfies these needs and provides related advantages as well. The present thermal cycler provides unprecedented cycling rates while retaining the usability of conventional PCR and real-time PCR platforms.